Oncogene (2013) 32, 4778–4788 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE The CDK1 inhibitory kinase MYT1 in DNA damage checkpoint recovery JPH Chow and RYC Poon Inhibition of cyclin-dependent kinase 1 (CDK1) by phosphorylation is a key regulatory mechanism for both the unperturbed cell cycle and the DNA damage checkpoint. Although both WEE1 and MYT1 can phosphorylate CDK1, little is known about the contribution of MYT1. We found that in contrast to WEE1, MYT1 was not important for the normal cell cycle or checkpoint activation. Time-lapse microscopy indicated that MYT1 did, however, have a rate-determining role during checkpoint recovery. Depletion of MYT1 induced precocious mitotic entry when the checkpoint was abrogated with inhibitors of either CHK1 or WEE1, indicating that MYT1 contributes to checkpoint recovery independently of WEE1. The acceleration of checkpoint recovery in MYT1-depleted cells was due to a lowering of threshold for CDK1 activation. The kinase activity of MYT1 was high during checkpoint activation and reduced during checkpoint recovery. Importantly, although depletion of MYT1 alone did not affect long- term cell growth, it potentiated with DNA damage to inhibit cell growth in clonogenic survival and tumor xenograft models. These results reveal the functions of MYT1 in checkpoint recovery and highlight the potential of MYT1 as a target for anti-cancer therapies. Oncogene (2013) 32, 4778–4788; doi:10.1038/onc.2012.504; published online 12 November 2012 Keywords: cell cycle; checkpoint; ionizing radiation INTRODUCTION CHK1/CHK2, which in turn inactivates CDC25 (reviewed in Chen 15 Cyclin-dependent kinase 1 (CDK1) is one of the major kinases for and Poon ). This response tips the balance of the CDK1 Thr14/ driving cells into mitosis. Its activation requires the binding of Tyr15 phosphorylation toward inactivation of the kinase. Indeed, cyclin B and the phosphorylation of the T-loop. Cyclin B starts to the checkpoint induced by ionizing radiation (IR) can be abrogated 16 accumulate from S phase and is destroyed at the end of mitosis.1 by overexpressing a non-phosphorylatable CDK1 mutant. Before mitosis, cyclin B–CDK1 complexes are inactivated through Once damaged DNA is repaired, the checkpoint has to be phosphorylation of Thr14 and Tyr15 by WEE1 and MYT1.2,3 While switched off to restart the cell cycle. This requires CDC25B, but not WEE1 specifically phosphorylates Tyr15, MYT1 shows a preference CDC25A and CDC25C, indicating a divergence of the roles of cell for Thr14. cycle components in checkpoint recovery and unperturbed cell 4 Just before mitosis, WEE1 and MYT1 are downregulated to allow cycle. Similarly, although PLK1 is not required for normal mitotic 4 CDK1 activation. The NH2-terminal region of WEE1 is phosphory- entry, it is important for checkpoint recovery. While WEE1 is lated by CDK1, PLK1 and CK2, creating a phosphodegron for known to have an important role in the G2 DNA damage 17 SCFbTrCP-dependent degradation.4–6 Any residual WEE1 is also checkpoint, next to nothing is known about the function of inhibited by CDK1- and AKT/PKB-dependent phosphorylation as MYT1 in the checkpoint. well as by binding to PIN1.7 By contrast, relatively little is known In this study, we have investigated the functions of MYT1 in about how MYT1 is inactivated. In mammalian cells, the checkpoint recovery. We found that while MYT1 was not essential inactivation of MYT1 coincides with its phosphorylation by PLK1 for the unperturbed cell cycle or the activation of the DNA damage and CDK1.2,8,9 checkpoint, it had a rate-determining function in checkpoint WEE1 has an indispensable role in controlling the timing of recovery. Downregulation of MYT1 also promoted DNA damage- mitosis during normal cell cycle. Depletion of WEE1 resulted in mediated cytotoxicity in cell line and nude mice models. premature chromosome condensation.10 By contrast, MYT1 appears to have a relatively minor role in the somatic cell cycle.11 As MYT1 is the only CDK1 inhibitory kinase in prophase- RESULTS arrested Xenopus oocytes, MYT1 is believed to have an important MYT1 is not involved in the control of the unperturbed cell cycle 12 role in the meiotic cycle during early development. Under this and activation of the G2 DNA damage checkpoint condition, MYT1 is inactivated by XRINGO- and CDK-mediated We started by analyzing the temporal expression pattern of MYT1 phosphorylation.13 In starfish, MYT1 activity is downregulated by in HeLa cells. Cells were released from a double thymidine AKT-dependent phosphorylation during meiosis.14 synchronization procedure and harvested every 3 h. Mitosis Proper cell cycle progression requires several checkpoints that occurred at between 9–12 h, as indicated by the DNA contents Tyr15 regulate the activities of CDKs. The G2 DNA damage checkpoint and the expression of phosphos-CDK and mitotic cyclins exerts its effect mainly through the inhibitory phosphorylation of (Figure 1a). While the expression of WEE1 decreased during CDK1. Upon DNA damage, ATM/ATR phosphorylates and activates mitosis, MYT1 displayed a dramatic gel mobility shift. Addition of Division of Life Science and Center for Cancer Research, Hong Kong University of Science and Technology, Kowloon, Hong Kong. Correspondence: Professor RYC Poon, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. E-mail: [email protected] Received 10 May 2012; revised 14 September 2012; accepted 18 September 2012; published online 12 November 2012 MYT1 in checkpoint recovery JPH Chow et al 4779 Figure 1. MYT1 is not important for the unperturbed cell cycle. (a) Level and phosphorylation of MYT1 during the cell cycle. HeLa cells were synchronized with a double thymidine block method. At different time points after released from the block, the cells were harvested and analyzed with flow cytometry (the positions of 2N and 4N DNA contents are indicated). Lysates were also prepared and the expression of the indicated proteins was analyzed with immunoblotting. Uniform loading of lysates was confirmed by immunoblotting for actin. The positions of the phosphorylated and unmodified MYT1 are indicated. (b) Gel mobility shift of MYT1 during mitosis is phosphorylation-dependent. MYT1 was immunoprecipitated from mitotic cell extracts (from cells collected at 12 h after release from double thymidine block as described in panel a). The immunoprecipitates were treated with either buffer or lPPase. The gel mobility of MYT1 was then analyzed with immunoblotting. (c) MYT1 is not required for the activation of various checkpoints. HeLa cells were either mock-transfected or transfected with siRNA against MYT1 (siMYT1). After 48 h, the cells were treated with hydroxyurea (HU), IR or nocodazole (NOC) for 16 h. Lysates were then prepared and the expression of the indicated proteins was analyzed with immunoblotting. Equal loading of lysates was confirmed by immunoblotting for actin. (d) MYT1 is not required for checkpoint-induced cell cycle arrest. Cells were transfected and treated as in panel c. The cells were then fixed and analyzed with flow cytometry. (e) MYT1 depletion does not affect the timing of mitotic entry. HeLa cells expressing histone H2B-GFP were transfected with siRNAs against MYT1, PLK1 or WEE1. The cells were synchronized with a double thymidine procedure and released into the cell cycle. Time-lapse microscopy was used to analyze the time taken by individual cells to enter mitosis (mean±90% confidence coefficient; n ¼ 30). The original data of the individual cells are shown in Supplementary Figure S2. the proteasome inhibitor MG132 stabilized WEE1 and cyclin A2 slower-migrating form of MYT1 after lambda phosphatase without affecting MYT1 (Supplementary Figure S1A), verifying that treatment verified that MYT1 was strongly phosphorylated during MYT1 was not regulated by degradation. The disappearance of the mitosis (Figure 1b).9 & 2013 Macmillan Publishers Limited Oncogene (2013) 4778 – 4788 MYT1 in checkpoint recovery JPH Chow et al 4780 To study the functions of MYT1, we depleted MYT1 with small indicate that unlike that for WEE1, there is no evidence that MYT1 interfering RNAs (siRNAs) (siMYT1) and verified the depletion with has a role in the timing of mitosis during the unperturbed cell immunoblotting (Figure 1c). This resulted in negligible effects on cycle of HeLa cells. normal cell cycle distribution (Figure 1d). MYT1-depleted cells were able to be trapped by hydroxyurea, IR or nocodazole in S phase, G2 phase or mitosis, respectively, indicating that the proper MYT1 is a component of the checkpoint recovery network activation of the various checkpoints did not require MYT1. Time- Several potential cell cycle regulators including PLK1 and CDC25B lapse microscopy also confirmed DNA damage was able to induce appear to be non-essential for normal G2–M transition, yet have a cell cycle arrest in MYT1-depleted cells (see below). important roles during the recovery of the G2 DNA damage WEE1 prevents premature activation of cyclin–CDK1 complexes checkpoint.19 To determine whether MYT1 is involved in during G2 phase. To evaluate the possible contribution of MYT1 on checkpoint recovery, siMYT1 was transfected before DNA the timing of mitosis, cells expressing histone H2B-GFP were damage was introduced with IR. HeLa cells were used as a generated and transfected with siMYT1. After they were model because IR exclusively activated the G2 DNA damage synchronously released from a double thymidine block, individual checkpoint in these cells (p53 is degraded by HPV E6). We used a cells were then tracked with time-lapse microscopy to monitor the CHK1 inhibitor (UCN-01) to turn off the checkpoint, thereby timing of mitosis (Supplementary Figure S2). The average time mimicking checkpoint recovery and inducing mitosis taken for the control cells to enter mitosis was B600 min synchronously in the population.20 Time-lapse microscopy (Figure 1e). This period was dramatically reduced to B300 min confirmed that almost no cell underwent mitosis after IR when WEE1 was downregulated with siRNA.